This invention relates to selective measurement of the number of different types of viable cells or their activity in a sample of cells through the utilization of surface active agents. This method is based on selective releasing of nucleotides from somatic cells and microbials cells, and subsequent measurement of nucleotides by bioluminescence.
Measurement of somatic (eucaryotic) and microbial cells is of great importance in medical laboratories, veterinary science, food hygiene, the fermentation industry and environmental sciences. Bacteria, yeasts and fungi are measured either by standard colony counting in a growth medium or by instruments, such as a microscope, turbidometer, nephelometer and the like. Somatic cells are counted with particle counters, such as an electro-optical particle counter and instruments based on detecting fluorescent particles, or indirect measurement based on the quantity of a metabolic product, or by microscopy. The methods require either complex, expensive equipment or are not accurate due to interference from non-cellular particles or non-somatic cells. Furthermore, most conventional methods do not make any distinction between dead and viable cells.
With conventional methods it has been difficult and time consuming to selectively determine different types of cells in samples containing both somatic and microbial cells. Conventional methods in microbiology have been so slow that it takes 24 to 72 hours to obtain the results. In clinical tests the results should be obtained as soon as possible in order to start proper treatment. In food hygiene it is as important to determine promptly the level of microbial contamination of raw materials and finished products, as in clinical samples, to protect the consumers and prevent spoilage of food. Therefore, rapid alternatives for colony counting have been looked for.
Firefly bioluminescent measurement of adenosine triphosphate (ATP) (see U.S. Pat. No. 3,745,090) is a rapid and sensitive method for determining the number of bacterial cells in a sample. In this method the quantity of measured ATP is converted to number of cells by dividing the quantity of ATP in the sample by the level of ATP per living cell. ATP level in a particular type of viable cell is relative constant. Similarly, the bioluminescent system of photobacteria has been used to measure bacterial cells by applying the level of assayed flavin mononucleotide (FMN) level in the sample to calculate the number of bacteria.
In order to determine the number of bacteria in foodstuffs and body fluids, such as milk, urine, blood, central spinal fluid, saliva and seminal fluid, ATP in somatic cells has to be eliminated quantitatively. Body fluids contain blood, epitelial, muscle and sperm cells, and the ATP level of these is higher than that of bacterial cells. Therefore, only a few somatic cells can cause a large error in the measurement of bacteria if somatic ATP is not eliminated prior to the microbial ATP assay. Likewise, the interference by the nucleotides from microbial cells has to be avoided when somatic cells are measured in samples containing both cell types. The utilization of bioluminescent assays has been limited by the lack of suitable simple and selective sample preparation methods for different types of cells.
Selective measurement of both somatic and microbial cells are needed in many fields of science, medicine, hygienic and industrial quality control. In these fields selective measurement of cells fall into three categories:
1. Measurement of somatic cells in the presence of microbial cells; PA1 2. Measurement of microbial cells in the presence of somatic cells; PA1 3. Measurement of total viable cells.
The present invention makes it possible to accomplish these measurements, rapidly, simply, and more accurately than with conventional methods. The principles and the different alternatives of utilization of the methods based on this invention are shown in the examples hereinafter described.